GSM6932273: T. brucei 14dpi mouse spleen, scRNAseq; Mus musculus; RNA-Seq
Single-cell suspensions were prepared from freshly collected mouse spleensby homogenizing spleens in 4 mL of Dulbecco's Modified Eagle Medium (DMEM) (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% Fetal Bovine Serum (FBS) (Atlas Biologicals, CO, USA) and 1% penicillin/streptomycin (Capricorn Scientific, Ebsdorfergrund, Gemany), using gentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). After passing the homogenate through a 70 µm cell strainer (SPL Life Sciences, Gyeongi-do, Korea), cells were centrifuged at 314 x g at 4oC for 7 minutes. Cell pellets were resuspended in RBC lysis buffer (Biolegend, CA, USA) at 4oC and incubated for 5 minutes. Cells were washed and re-suspended in PBS supplemented with 0.04% BSA. Total remaining live cells were counted by Trypan Blue (Sigma-Aldrich, Missouri, USA). Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, splenocytes were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. were loaded into single cell chips (10X Genomics) and partitioned using Gel Bead In-Emulsion (GEM) technology and a Chromium Controller (10X Genomics). Single cell libraries were prepared using the 10X Genomics Chromium Single Cell 3`reagent kit v3. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.